1. Volume 35, Issue 4, Pages 657-669 (August 2002)
Neural Crest Stem Cells Persist in the Adult Gut but Undergo Changes in Self-Renewal, Neuronal Subtype Potential, and Factor Responsiveness 
Genevieve M. Kruger, Jack T. Mosher, Suzanne Bixby, Nancy Joseph, Toshihide Iwashita, Sean J. Morrison 
Neuron 
Volume 35, Issue 4, Pages (August 2002)
DOI: /S (02)

2. Figure 1 Multipotent Neural Progenitors Persist in the Postnatal Gut
Cells were dissociated from P15 gut and cultured at clonal density (40 cells/35 mm dish) for 14 days, then stained for neurons (Peripherin, [C]), glia (glial fibrillary acidic protein, red in [B]), and myofibroblasts (smooth muscle actin, green in [B]). One field of view from a typical multilineage colony is shown (A–C). Freshly dissociated P15 gut cells were also analyzed by flow-cytometry for expression of the neurotrophin receptor p75 and α4 integrin (D). Cells in the regions marked on the flow-cytometry plot shown in (D) were sorted into clonal culture for analysis. Multipotent progenitors were enriched in the p75+ fraction but depleted in other fractions (see Results).
Neuron  , DOI: ( /S (02) )

3. Figure 2 Multipotent Neural Crest Progenitors Are Present in Adult Gut
One field of view from a typical colony cultured from adult rat gut was stained for neurons (Peripherin; [C]), glia (GFAP; [B]), and myofibroblasts (SMA; [A]) as described in Figure 1. DAPI was used to label all cell nuclei (blue in [B]). Multipotent adult colonies were estimated to average 20,000 ± 7000 total cells/colony and 500 ± 770 neurons/colony after 14 days of culture.
Neuron  , DOI: ( /S (02) )

4. Figure 3
Postnatal Gut NCSCs Are Significantly Less Mitotically Active Than Embryonic Gut NCSCs
Cells were stained with antibodies against p75 and α4 integrin to identify E14.5 and P15 gut NCSCs, and the amount of DNA per cell was quantitated by flow-cytometry using the DNA-intercalating dye Hoechst (Morrison and Weissman, 1994). The percentage of E14.5 (A) and P15 (B) gut NCSCs in G0/G1 (2N DNA content) and S/G2/M (>2N DNA content) phases of the cell cycle was determined. To compare the rate at which NCSCs divide in vivo, BrdU was administered to rats for a 20 hr period in vivo prior to isolating and assaying p75+α4+ E14.5 gut NCSCs or p75+ P15 gut NCSCs for BrdU incorporation. An average of 87% of E14.5 NCSCs (but only 13% of P15 gut NCSCs) incorporated BrdU and therefore divided over this period in vivo. These data indicate that E14.5 gut NCSCs divide more rapidly or are less frequently quiescent than P15 gut NCSCs.
Neuron  , DOI: ( /S (02) )

5. Figure 4 P15 Gut NCSCs Give Rise to Neurons and Glia In Vivo
Uncultured P15 rat gut p75+ cells were isolated by flow-cytometry and injected into two hindlimb bud somites of stage 18 chick embryos. After approximately 72 hr of development (stage 29), embryos were fixed and frozen and then sectioned at the level of the hindlimb. Sections were hybridized in situ with probes against the neuronal marker SCG10 and the glial marker P0. Hybridization with neuron-specific probes is shown in (A), where the purple signal indicates SCG10+ rat neurons (arrowheads) and the orange signal indicates chick neurons (arrows) in a sympathetic ganglion. Hybridization with a rat-specific probe against the glial marker P0 ([B], open arrowheads) showed rat glia in chick peripheral nerves. Both (A) and (B) are from the same embryo.
Neuron  , DOI: ( /S (02) )

6. Figure 5
p75+ NCSCs Give Rise to Neurons in Culture that Express a Variety of Neurotransmitters Normally Found in the Enteric Nervous System
Gut p75+ cells were cultured under standard conditions for 14 days and then stained with antibodies against neuronal Nitric Oxide Synthase (nNOS) (A), Neuropeptide Y (NPY) (C), or Vasoactive Intestinal Peptide (VIP) (B). nNOS, NPY, and VIP were present within neuronal cell bodies and neurites as expected. (A)–(C) are photos taken of adult gut NCSC colonies. Nearly all multilineage colonies formed by E14.5 p75+α4+ NCSCs and P15 and adult gut p75+ NCSCs contained nNOS+ cells, NPY+ cells, and VIP+ cells with neuronal morphologies (D). No differences were detected between E14.5 and P15 gut NCSCs in either the frequency of colonies containing cells that stained with these markers (D) or in the frequency of cells within colonies that stained with these markers, though adult colonies appeared to contain a lower density of nNOS+ cells (data not shown).
Neuron  , DOI: ( /S (02) )

7. Figure 6
E14.5, But Not P15, Gut NCSCs Generate Serotonergic Neurons in Culture
E14.5, P5, and P15 gut NCSCs were cultured under standard conditions for 14 days and stained for the presence of serotonergic neurons using an antibody against Tryptophan Hydroxylase. Serotonergic neurons were present at low density in the majority of multilineage E14.5 gut colonies (arrow in [A]) but were not detected in P15 gut multilineage colonies (B). The decline with age in the percentage of multilineage colonies containing any serotonergic neurons is shown in (C). A significantly higher fraction of E14.5 gut multilineage colonies contained serotonergic neurons than did either P5 or P15 gut colonies (p < 0.001).
Neuron  , DOI: ( /S (02) )

8. Figure 7
Postnatal Gut NCSCs Decline in Their Ability to Form Tyrosine Hydroxylase-Expressing Neurons with Increasing Age
NCSCs from E14.5, P15, and P22 gut were cultured at clonal density under conditions that support the differentiation of noradrenergic neurons (Morrison et al., 2000a). One field of view from a typical E14.5 gut NCSC colony (A, D, and G), P15 NCSC colony (B, E, and H), or P22 NCSC colony (C, F, and I) is shown in phase contrast (A–C), with Peripherin epifluorescence (D–F), or with tyrosine hydroxylase (TH) epifluorescence (G–I). Double labeling of similar cultures with Peripherin and Dopamine-β-hydroxylase (DβH) is shown in Supplemental Figure S2. In both cases, the number of neurons per colony that expressed TH or DβH declined significantly with increasing NCSC age. Among colonies that contained TH+ cells, E14.5 gut NCSC colonies averaged 307 ± 244 TH+ neurons/colony, P5 gut NCSC colonies averaged 11 ± 10 TH+ neurons/colony, P10 NCSC colonies averaged 7 ± 10 TH+ neurons/colony, and P15 gut colonies had only 2 ± 1. Typical neurons that costained for TH and Peripherin are shown with white arrows (D and G), and a neuron labeled with Peripherin, but not TH, is shown with a yellow arrow (D and G). Note that TH+ cells tended to be clustered within part of each colony, so although most Peripherin+ neurons appear TH+ in (D) and (G), only a minority of Peripherin+ neurons within the overall colony were TH+.
Neuron  , DOI: ( /S (02) )

9. Figure 8
Gut NCSCs Become Increasingly Responsive to the Gliogenic Effect of Soluble Delta as Development Proceeds
Freshly isolated NCSCs were cultured for 24 hr at clonal density (40 cells/well) in standard medium (no add) or standard medium supplemented with either Fc or Delta-Fc (Morrison et al., 2000b). These factors were then washed out of the cultures, and the cells were cultured under standard conditions for another 13 days. There were no significant differences between NCSCs cultured in no add and Fc only conditions. Delta treatment did not significantly increase the frequency of glial only colonies from E14 gut NCSCs, but it did significantly increase the frequency of glial only colonies from P5 and P15 gut (p < 0.05). 29%–43% of E14 cells, 45%–67% of P5 cells, and 54%–67% of P15 gut cells survived to form colonies in culture. The data are based on three independent experiments.
Neuron  , DOI: ( /S (02) )